ES2932185T3 - Optimized volumetric imaging with selective volume illumination and light field detection - Google Patents

Abstract

Systems and techniques related to optimizing volumetric imaging with selective volume illumination (SVI) using light field detection, in one aspect, include: a light source configured to emit an illumination light that propagates through a illumination light path to illuminate a three-dimensional (3D) sample object; and an optical system arranged with respect to the light source to receive a light field, which comes from the illuminated 3D sample, in which the light field propagates through a detection light path; wherein the light source, the optical system, or both, are configurable to select a volume of a 3D confined illumination of the 3D sample based on the 3D sample to be illuminated and a light field detection (LFD) process to apply. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Optimized volumetric imaging with selective volume illumination and light field detection

Antecedent

This specification is concerned with optimizing volumetric imaging using light field detection with biomolecule imaging systems, such as microscopy platforms.

Various biological procedures may depend on the close spatiotemporal coordination between cells through three-dimensional (3D) space at the tissue scale. Therefore, there is a need for volumetric imaging tools that can image with cellular resolution, provide 3D slices, and at speeds fast enough to accurately capture dynamic procedures. In some cases, the need for biological volumetric imaging is met with scanning approaches: the optical signal is collected one point, line, or plane at a time, and then the excitation region is scanned in 3D, 2D, or 1D, respectively, to cover the entire 3D region of interest of the sample. Due to the sequential nature of the scanning procedure, the data acquisition speed can be slow. Also, due to sequential scanning, information from different parts of the 3D sample is collected at different times, therefore dynamic information may be lost or distorted. Therefore, there is a need for a synchronous in vivo volumetric imaging modality, in which a single snapshot can capture spatial information about an axially extended volume at the tissue scale, while maintaining a resolution of one or few times. cells.

An existing imaging modality that can provide synchronous volumetric imaging capability is light field microscopy (LFM). However, LFM techniques use wide-field illumination, which essentially illuminates the entire sample, even though only a single part of the sample is significantly captured in the reconstruction. Therefore, wide-field illumination introduces extraneous background and noise, which can potentially degrade the contrast and accuracy of the final reconstructed images.

Noy Cohen describes in Optical Express, Volume 22, Number 20, October 3, 2014 on page 24817 improving light field microscope performance using wavefront coding.

US 2012/200829 A1 describes an imaging device which may include at least one single pixel imaging sensor configured to detect image data for a single pixel along a line of sight. The imaging device may also include at least one reorientable mirror, each reorientable mirror of which is uniquely optically coupled to one or more of said at least one single-pixel imaging sensor for deflecting line of sight. of that single-pixel imaging sensor. The imaging device may further include a controller for synchronously reorienting each of said at least one reorientable mirror to scan the line of sight through at least one sector of a scene. The imaging device may also include a readout circuit for reading image data acquired from each of said at least one single-pixel imaging sensor. The imaging device may be configured to sample at least two independent dimensions of a four-dimensional field of light. A plenoptic imaging device and a plenoptic projector device are also disclosed.

Document US 2015/381908 A1 describes an imaging system comprising a light field camera (3) for recording a hyperspectral light field (CLF). The system also comprises a light projector (4) to project a field of light in visible light (PLF). The camera and the projector share a common optical axis. The projector projects a field (PLF) of light based on the field (CLF) of hyperspectral light captured by the light field camera.

Summary

This specification relates to systems and techniques for optimizing volumetric imaging using Selective Volume Illumination (SVI) with light field detection used in biomedical imaging systems, such as microscopy platforms.

In general, one or more aspects of the subject matter described herein may be incorporated into one or more systems including: a light source configured to emit illuminating light propagating through a light path lighting to illuminate a three-dimensional (3D) sample; and an optical system arranged with respect to the light source to receive a light field, which comes from the illuminated 3D sample, wherein the light field propagates through a detection light path; in which the source light, the optical system, or both, are configurable to select a volume of a 3D confined illumination of the 3D specimen based on the 3D specimen to be illuminated and a light field detection (LFD) procedure. ) to apply.

Particular embodiments of the subject matter described herein may be implemented to realize one or more of the following advantages. The described systems and techniques can realize advantages associated with the SVI ratio where only the relevant part of the 3D sample is illuminated, thereby significantly reducing background signal and providing higher contrast and accuracy for image reconstruction. of light field. The disclosed techniques implement a scanned optical volume SVI that provides precision down to the micrometer level in defining the SVI. A static optical volume SVI reduces the likelihood of photoinduced damage to the biological sample during imaging.

The disclosed techniques and systems implement Selective Volume Illumination Light Field Detection (SVI-LFD) imaging capabilities within the framework of a conventional microscopy rig, using the same setup, with the same sample under study. , to record a high-resolution 3D image, optically sectioned (using conventional microscopy mode), in addition to the volumetric image reconstructed by light field. SVI approaches can improve the speed and accuracy of computational reconstruction used in light field imaging. SVI techniques use smaller, more constrained excitation volumes, allowing faster, less noisy, and more robust computational reconstruction. A multi-view light field detection (LFD) imaging technique is implemented which performs improved resolution of a final volumetric image by combining image data captured from sample imaging from multiple directions.

Details of one or more embodiments of the subject matter described herein are set forth in the accompanying drawings and description below. Other features, aspects, and advantages of the invention will be apparent from the description, drawings, and claims.

Drawing Descriptions

Figure 1 shows an example of a volumetric imaging system used to provide Selective Volume Illumination (SVI) using light field detection.

Figures 2A-2C show examples of general optical geometries for implementing the SVI techniques described herein, applicable to various static optical volume and scanned optical volume approaches.

Figure 2D shows an example of an optical geometry for implementing the SVI techniques described herein, according to a scanned optical volume approach.

Figure 3A shows an example of a multicore fiber for use with an endoscopic implementation of SVI-LFD imaging techniques.

Figures 3B-3F show examples of optical geometries for use with endoscopic implementations of SVI-LFD imaging techniques.

Figures 4A-4C show examples of optical geometries for use with an implementation of SVI using multiple view LFD imaging techniques.

Similar reference numbers and designations in the various drawings indicate similar elements.

Detailed description

Figure 1 shows an example of a volumetric imaging system 100 used to provide Selective Volume Illumination (SVI) using light field detection. For example, volumetric imaging system 100 provides light field capabilities in support of 3D reconstruction of a microscope specimen 126. According to embodiments, volumetric imaging system 100 may be used to provide images in 3D at cellular resolution or better, and 3D sectioning, at speeds that can capture dynamic procedures (eg, low-resolution/high-speed volumetrics). As illustrated in Figure 1, the system integrates various hardware, software, and firmware components that work together to achieve the embodiments' volume-selective illumination techniques, so as to illuminate a 3D confined illumination of the specimen 126 , which is a particular portion of the 3D sample that has been determined to be substantially relevant for imaging volumetric. That is, the 3D confined illumination is a selected volume of the 3D sample 126 to be particularly excited by an illumination light for imaging.

The volumetric imaging system 100 shown in Figure 1 includes: a volumetric imaging apparatus 105; an LFD mechanism 115; a network 135; and a computer 140. Volumetric imaging apparatus 105 includes various system components 100 arranged to emit light to illuminate a volume of the 3D sample 126 to be obtained. In some embodiments, volumetric imaging system 100 is implemented to provide light field images of enhanced contrast agents. For example, the photochemical and physical properties of the contrast agents in sample 126 can be changed by exposure to an illumination light, offering enhanced contrast in a selective 3D volume of sample 126. Once the properties of Since contrast agents have been modified into a selective volume, high-contrast LFD imaging of that volume can be achieved even with conventional wide-field illumination. Contrast agents may include: photoactivated or photoconvertible fluorescent proteins, or photoactivated or photoconvertible synthetic particles, such as, but not limited to, nanoparticles or quantum dots.

As illustrated, the volumetric imaging apparatus 105 includes: a light source 106; an optical system 110; and a microscope assembly 125. The optical system 110 includes one or more optical elements arranged to adjust the optical geometry of the apparatus 105 to provide optical paths to the 3D sample 126 and detection mechanism 115 . As an example, the optical system 110 is shown to include various optical elements, such as one or more lenses 112, and mirrors 114. However, various components of the optical system 110 may be changed, or otherwise configured, as deemed necessary and/or or appropriate to implement an optical geometry corresponding to the desired SVI-LFD imaging technique(s). In some embodiments, optical system 110 is a plug-in module that can be adapted to microscope assembly 125, for example, including switchable optical components and optical geometries. Furthermore, in some implementations, microscope assembly 125 may be originally designed and manufactured with SVI-LFD capabilities built into it, and including all necessary components, such as optical system 110. Additional lenses and/or other optical elements can be combined to form an objective lens, for example, from system 100. These elements can be installed, for example, by mounting to a section of microscope assembly 125. In addition, an SVI light arm can be added to the microscope assembly 125. For example, the SVI light arm can be attached to a spot scanning confocal microscope via a partially reflective beam splitter or dichroic mirror (for illuminating or sensing lights that are of different colors).

The volumetric imaging system 100 may include a volumetric imaging apparatus 105 that is capable of being configured to implement the described SVI-LFD imaging techniques so as to significantly reduce signal background compared to with some existing volumetric imaging technologies operating in a manner which illuminates the entire sample. Therefore, the volumetric imaging system 100 can be used to restrict the illumination of the light source 106 to a selected volume of a 3D confined illumination of the specimen 126, which can be any volume that is less than the entire of the sample 126 in 3D. Therefore, the system 100 realizes a solution that provides higher contrast and accuracy of the light field image reconstruction by means of the light field detection (LFD) mechanism 115 .

Microscope assembly 125 may be a microscopy platform configured to implement existing microscopy imaging capabilities. For example, microscope assembly 125 is a spot scanning confocal microscope capable of high resolution 3D optical sectioning in accordance with some existing microscopy imaging modalities. The high-resolution image data collected through the high-resolution imaging capability of the microscope assembly 125 can be used to improve the speed, resolution, contrast, or accuracy of image reconstruction of image data. SVI-LFD image. In some cases, optical sectioning is performed with a resolution of submicrometers to 2 micrometers. In some implementations, the microscope assembly 125 is a light sheet microscope. The microscopy functions associated with the microscope assembly 125 may use light beams that have different propagation properties (eg, width, divergence, cross-sectional area, beam shape) than the light beams used in the acquisition functions. of SVI-LFD images. The generation of an illumination light that can be suitably employed by each of the imaging modes, respectively, can be implemented using optical components such as cylindrical lenses, spherical lenses, engineering diffusers, prisms, and the like.

Therefore, the volumetric imaging system 100 is usable for recording high resolution 3D images, or structural images, in addition to light field reconstructed 3D images having lower resolution, or light field images. In some cases, the structural images obtained using the microscopy capabilities of the system 100 are used to augment the computational reconstruction of the structures. light field images. Conceptually, since both images are of the same sample region, potentially separated by short times between image capture, information regarding the sample volume of interest can be obtained from both imaging modes. Consequently, the high-resolution and information-rich content of structural images can be used to guide the reconstruction of SVI-LFD images. In some cases, the use of structural imaging in conjunction with light field reconstruction may provide advantages such as speeding up reconstruction processing (for example, providing a convergent solution more quickly), and/or constraining the reconstruction to obtain more accurate results. precise.

In accordance with SVI techniques, the light source 106 can be implemented as illumination mechanisms usable with existing microscopy platforms. Continuous wave lasers for linear signal contrast, and pulsed lasers for non-linear signal contrast can also be used as the light source 106 . Additionally or alternatively, SVI techniques can be implemented with non-coherent or low-coherence light, such as light-emitting diodes (LEDs). In the case of using non-coherent mechanisms for the light source 106, the SVI illuminates an extended volume. In some implementations, the light source 106 is a coherent light mechanism. In this case, specific spatial regions of constructive and destructive light interference that are achieved by using coherent illumination light can implement the SVI aspects of light field imaging. Examples of coherent light mechanisms used to implement light source 106 may include holography, interferometry, and the like. In addition, a light source 106 may be used to produce illumination light that has the necessary properties to change the photochemical and physical properties of the contrast agent(s) in the sample, providing enhanced contrast in a selective 3D volume of the sample. sample. In some implementations, one or more additional light sources may be used in conjunction with light source 106. For example, an additional light source can be used to provide light field imaging of contrast enhancing agents.

The light field detection mechanism 115 may be used to detect and generate image data based on the light field detection techniques. Furthermore, in one implementation, the LFD detection mechanism 115 may include a camera including optics that image in the plane of an array of microlenses that can redirect, for example, the light field to a sensor. In some implementations, the LFD mechanism 115 can generate LFD images as digital information displaying the resulting image directly on the computer screen 145. The LFD mechanism 115 may employ other forms of imaging and light field detection as necessary or appropriate. By incorporating the SVI-LFD imaging capabilities into the microscope assembly 125, additional LFD-based imaging components can be designed as add-on modules to a microscopy platform as the LFD mechanism 115 . As an example, a camera detection path may be added to the optical geometry of the apparatus, and a camera capable of capturing LFD images is included in the LFD mechanism 115.

In addition, the SVI techniques disclosed herein can be used in conjunction with other LFD technologies, including, but not limited to: wavefront coding with a phase mask; spatial light modulator, or similar devices; multicolor detection through the use of dichroic and bandpass filters; various ways of manufacturing and implementing the microlens array, including, but not limited to, the use of electrically tunable oil droplets for the lenses, or non-uniform or shaped microlens arrays; and the like.

Computer 140 includes a processor 142 and memory 144, and computer 140 may be connected to a computer network 135, which may be a private network, a public network, a virtual private network, etc. Computer network 135 can be implemented as a wired network (eg, Ethernet) or a wireless network. The various devices of system 100, including volumetric imaging apparatus 105 and computer 140, may be connected via network 135. Some examples of computer networks include, but are not limited to, a local area network (LAN), a wide area network (WAN), and the Internet. As shown in Figure 1, the computer network 135 is a wired network, so that the volumetric imaging apparatus 105 can communicate, or otherwise transmit, data through an interface to one or more computer components. using physical connections, such as Universal Serial Bus (USB) connectors. Additionally or alternatively, the devices may use a direct physical connection that need not be used by network 135. In some implementations, computer network 135 may be a wireless local area network (WLAN), such as an IEEE 802 network. no. Thus, the volumetric imaging apparatus 105 and the computer 140, for example, are communicatively coupled, and are capable of wirelessly transmitting data over the computer network 135.

Processor 142 may be one or more hardware processors, which may each include multiple processor cores. Memory 144 can include both volatile memory and non-volatile memory, such as Random Access Memory (RAM) and Flash memory. Computer 140 may include various types of computer storage media and devices, which may include memory 144, for storing program instructions running on processor 142.

Such programs may include SVI-LFD Imaging Software 146, which may run locally on computer 140, remotely on a computer of one or more remote computer systems (for example, on the server system). a third party provider accessible by computer 140 via network 135), or in a combination of one or more of each of the above. The SVI-LFD Imaging Software 146 may present a user interface (UI) used to display, or otherwise provide, a reconstructed volumetric image 130 of the sample on a computer 140 display device 145. The device Display 145 may be operated using one or more input devices 148 of computer 140 (eg, keyboard and mouse or touch screen). It should be noted that, although shown as separate devices in Figure 1, display device 145 and/or input devices 148 may also be integrated with each other and/or with computer 140, such as in a tablet computer. .

The SVI-LFD Imaging Software 146 is configured to analyze, process, and manipulate volumetric imaging data associated with the sample, which is generated by the SVI techniques of the embodiments. The SVI-LFD Imaging Software 146 can implement various aspects of the SVI used to select and perform 3D volume constrained analysis of the 3D sample. As an example, for a given volume of interest, the SVI-LFD Imaging Software 146 is configured to determine multiple regions of interest, and command and/or control the various components necessary to perform excitation and acquisition. of images of the regions in a sequential manner according to the techniques. As another example, the SVI-LFD Imaging Software 146 may be implemented to automatically calculate, select, or control other facets of the SVI, such as proper SVI focus (eg, SVI static optical volume). , the adjustment of the optical geometry, the selection of the excitation mode, etc. In addition, the SVI-LFD Imaging Software 146 implements various aspects of the LFD imaging capabilities of the system 100. In some implementations, the SVI-LFD Imaging Software 146 is configured to generate data from obtaining volumetric images of the sample in 3D by performing a computational reconstruction of the LFD images. In some implementations, the SVI-LFD Imaging Software 146 is programmed to control the illumination of a volume of interest, and to effect movement of any electromechanical components necessary to properly adjust the optics to perform SVI-LFD operations ( for example, scanning). For example, SVI-LFD Imaging Software 146 can cause illumination of the 3D sample in a patterned or structured manner. The combination of patterned and/or structured illumination can result in a detected light field covering the desired 3D space of the sample, and allow for optimized computational reconstruction of the final image data captured from the light fields. light detected.

Figures 2A-2C show examples of optical geometries for implementing the SVI techniques described herein, according to various static optical volume approaches. In some cases, static optical volume approaches to implement SVI potentially provide additional advantages compared to other approaches described (shown in Figures 3A-3E and Figures 4A-4C). For example, in scenarios that involve imaging biological samples with larger volumes, performance trade-offs in accuracy (for example, defining volume limits) may be made in favor of ease of implementation. compared to other SVI approaches (eg, fewer optics than in endoscopic and multiple-view approaches), and decreased potential photoinduced damage to the specimen.

It is common among the optical geometries for the static optical volume approaches of Figure 2A-2C that the illuminating light can be formed using a direct light beam. In the present implementation, a direct beam of light is used to shape the illumination light, and produce a spatially extended volume of light that fills the desired volume of interest, i.e., the 3D confined illumination of the sample 205 in 3D. Figure 2A shows sample 205 as a biological specimen to be volumetrically visualized according to SVI-LFD techniques. In some implementations, propagation invariant optical beams may be used, such as Bessel or Airy beams. These types of beams can be used to gain the potential advantage of greater axial spread for a given beam waist, which improves precision in excitation volume illumination and results in more accurate 3D confined illumination.

Figure 2A shows a static optical volume approach which employs dedicated optics for illumination light 215 for excitation of sample 205, and light field 225 which comes from sample 205, respectively, in order to achieve the SVI aspects of volumetric imaging. As illustrated, illumination light 215 is emitted onto sample 205 through a lens 220 that is different from the lens 230 used to propagate the light field 225 . In some implementations, the lens 220 used for illumination is a microscope objective lens. In some implementations, the lens 230 used for detection is a detection objective lens. Therefore, the sample 205 can be illuminated via an illumination light path, in which the illumination light propagates, which is separated from the light detection light path 215, in which the field propagates. of light emanating from sample 205. Further, it is illustrated that the light detection light path for light field 225 is not collinear to the illumination light path for illumination light 215. That is to say, the light paths traveled for illumination and detection do not follow the same line. Figure 2A shows an illumination optical axis that is at an angle 0 theta from a detection optical axis. In accordance with the techniques described, this non-collinear nature of the illumination light path, for illumination light 215, and the light detection light path, for light field 225, allow for 3D confined illumination of the image. displays 205 in 3D, and thus achieve the SVI. In some cases, the optical geometry is adjusted such that the (0)theta angle is 90° in order to achieve SVI. In some embodiments, it is sufficient to perform the SVI by adjusting the corresponding optical geometry such that it sets the light-detection light path and the light-detection light path to any one of a range of angles for 0 which is greater than zero. . By employing an axis of illumination, with respect to the focal plane of the lens 220 that is not collinear to the detection axis, with respect to the focal plane of the lens 230, the illumination of the sample 205 is restricted to a 3D volume of interest, thus minimizing signal background that can be detrimental to the final contrast and computational reconstruction accuracy of light field imaging techniques.

Figure 2B illustrates another implementation for static optical volume focusing, in which the optical geometry is arranged to emit illumination light 235 through the same lens that is also used for detection, ie, lens 220. In some implementations, lens 220 is a microscope objective lens. The light field 240 which comes from the sample 205 propagates through the lens 220. Therefore, as illustrated, the illumination light path and the light field detection path cross through the lens. 220 sole. To achieve SVI according to the optical geometry shown in Figure 2B, illumination light is sent through decentered lens 220. In some implementations, illumination light 235 is delivered, from the light source, for example, at an oblique angle to the image plane of sample 205. The detection light path is collinear with the optical axis of the common optical element 220, making an angle (0) theta that is greater than zero with the illuminating light path. The oblique angle nature of the detection path allows multiple detection paths to be performed simultaneously, or sequentially, through the same microscope objective, without the need to physically rotate the sample. Consequently, even though a common lens 220 is shared for illumination light 235 and light field 240, the illumination and detection paths remain non-collinear in a manner similar to that shown in Figure 2A. Therefore, using the oblique angle illumination strategy as part of the optical geometry yields the SVI.

In some implementations, adjusting the corresponding optical geometry such that it arranges the illumination light path at any one of a range of angles that is both greater than zero, and not 90°, is sufficient to create a path of oblique illumination light and optimize excitation to the volume of 3D confined illumination of specimen 205. In some cases, the oblique angle illumination approach of Figure 2B can be used to implement aspects of SVI for imaging performed by a microscope assembly (shown in Figure 1) in addition to some existing microscopy capabilities of the microscope, such as raster point confocal laser scanning. In some cases, this implementation uses the single-photon excitation mode and switchable optical geometries to support, for example, operation in the SVI-LDF imaging mode or in the confocal microscopy mode. Implementations involving the use of SVI-LFD-based images with additional images, captured using another mode of microscopy, are discussed in detail with reference to Figure 1.

The static optical volume approaches of Figures 2A-2B can employ various modes of linear excitation to capture optical signal contrast. Linear excitation modes may include, but are not limited to: single-photon excitation fluorescence; elastic light scattering; and inelastic light scattering (eg, Raman or Brillouin scattering). In some implementations, where signal contrast is through elastic scattering of illumination light, techniques such as Raleigh or Mie scattering, or combinations of these, provide a label-free procedure for observing structures and procedures. biological. In cases where the SVI signal contrast is through inelastic scattering of illumination light, the signal contrast is label free and provides spectroscopic information about the sample. By employing a linear drive, a signal intensity can be generated that is proportional to the first power of the illumination light intensity, thus providing the necessary spatial constraint to obtain the SVI.

Figure 2C represents another example of an optical geometry used to implement the static optical volume approach for SVI techniques, using a shared optical element for illumination and detection, similar to the optical geometry shown in Figure 2B. . However, as illustrated in Figure 2C, the illuminating light 250 and the light field 255 emanating from the sample 205 propagate collinearly with respect to each other. That is, the light path and the light field detection path are aligned along the same axis, instead of being arranged non-collinearly as in the static optical approaches discussed above (shown in Figures 2A). -2B). In the collinear optical geometry, to avoid the similar function of some existing microscopy approaches (for example, wide-field illumination) and their drawbacks, multi-photon nonlinear excitation is used.

In order to achieve the described SVI techniques, the optical geometry of Figure 2C also allows the use of non-linear modes of optical excitation to generate signal contrast. For example, multi-photon excitation includes the interaction of the sample 205 with two or more photons nearly simultaneously to produce the contrast of the optical signal. These signal contrasts may include two-photon excited fluorescence, second harmonic generation, three-photon excited fluorescence, and higher order procedures. In these multi-photon modes, the signal intensity is proportional to the second (or third) power of the excitation intensity, for procedures involving two (or three) excitation photons, leading to a natural confinement of the excitation volume in 3D space, in cases where a focused beam is used to illuminate the sample (shown in Figure 2D). This nonlinear excitation confinement can be exploited as a key working principle of implementation of SVI approaches, in addition to some existing capabilities of multiphoton raster point laser scanning microscopes.

Furthermore, the static optical volume approaches corresponding to the optical geometries shown in Figures 2A and 2B also work with nonlinear excitation. With the optical geometry illustrated in Figure 2C, even with illumination light 250 and light field 255 arranged collinearly, the use of multi-photon nonlinear optical excitation will provide efficient SVI due to the nonlinear confinement of the excitement. In some implementations, the illumination light 250 and the light field 255 are received through the same microscope objective lens 260 . Also, in some cases, the signal mode is implemented as multi-photon excited fluorescence (eg, two-photon fluorescence, second harmonic generation, three-photon excited fluorescence). Due to the non-linear confinement of the signal, each of the static optical volume approaches to SVI described herein should provide efficient SVI. The first example shown in Figure 2C, in some cases, can be used to implement the combination of SVI-LFD techniques with some conventional microscopy techniques, such as multi-photon fluorescence microscopy.

Figure 2D shows an example of an optical geometry for implementing the SVI techniques described herein, according to a scanned optical volume approach. According to this technique, SVI is achieved because the illumination light 265 is a focused beam of light that is used to selectively scan, or "paint," the sample 270 volume of interest, rather than illuminate the entire sample. . Depending on the size and shape of the focal region, the excitation volume is "painted" (ie, filled in) by scanning in one, two, or three dimensions. It is important to note that all the different manifestations of the static optical volume approach described above are amenable to the scanned optical volume approach. Furthermore, in the scanning approach, the detection optics for capturing the light field image, such as a detection camera, is exposed for the time necessary to scan the entire confined volume in 3D. Therefore, one exposure time is sufficient to scan the entire sample volume of interest, avoiding the need for fragmentary scanning of segments of the volume of interest using multiple exposure times (for example, one exposure time for each segment to be scanned). In cases where fast scanning is implemented, eg scanning hardware with high rotation speeds, multiple rounds of scanning of the specified volume can be performed in a single exposure time. Performing SVI according to scanned optical volume techniques can provide multiple advantages, such as higher precision (eg, down to the micrometer level) in defining the SVI. Therefore, SVI scanning implementations may be desirable in scenarios where the design trade-offs of using more sophisticated hardware, compared to static optical volume approaches (shown in Figures 2A-2C), are tolerable for more accurate images.

As illustrated, a scanned light sheet 275 can be formed by shaping the illumination light 265 into a 2D static light sheet, using various optical elements arranged to focus the light, such as cylindrical lenses. The sheet of light 275 can then be scanned in a third dimension, to optimize illumination of the 3D confined illumination volume of the sample. In some cases, scanning is implemented by scanning the focal region using rotation/translation devices included in the optical setup, such as galvanometers or resonant scanners, or acousto-optic devices, such as acousto-optic baffles.

In some cases, an optical geometry for scanned optical volume focusing adjusts the illuminating light 265 (shown along the x-axis) at an angle 0 theta that is greater than zero with respect to the field detection direction 280 . of light (shown along the z-axis). Therefore, the illumination light 265, which is a focused beam of light, will appear as a nominal line within the field of view defined by the detection optics. Next, to achieve the SVI of a 3D volume, the illuminating light can be scanned in two dimensions.

The scanning approach can implement SVI with nonlinear excitation using multiple optical configurations, even in cases with collinear sensing and illumination paths (shown in Figure 2C), due to the confinement of signal contrast generation to only the focused region. To improve coverage and reduce photoinduced biological damage, often associated with high light intensities, low numerical aperture focused excitation beams can be used, as shown in Figure 1C, to have extended axial extension in the focal region. Then the 2D scan of the focus point will automatically provide a 3D excitation volume, achieving SVI. It should be noted that the larger cross-sectional area of the low numerical aperture focused beams will have a minimal effect on the final achieved resolution of the SVI-LFD, since the resolution in this case does not depend on the size of the illumination beam, but of the general optical properties of the LFD arm (numerical aperture of detection, focal length and aperture of the tube lens, lens array parameters, camera pixel size, etc.).

Figure 3A shows an example of a multicore fiber 305a for use with an endoscopic implementation of SVI-LFD imaging techniques. For example, an endoscope is integrated into the volumetric imaging system (shown in Figure 1) to provide video imaging capabilities while applying SVI-LFD imaging techniques. Implementations involving endoscopy may provide the advantages associated with performing SVI-LFD imaging remotely, or in locations that would otherwise be inaccessible to some microscopy-based imaging techniques. Figure 3A illustrates a multicore fiber 305a for supplying illumination light for excitation of a sample, or for transmitting detected light along the detection light path, whenever an image is to be transmitted, according with endoscopic executions. In addition, a single core fiber can be used for the illumination or detection light path, whenever it is necessary to transmit non-imaging light along the light paths. Therefore, SVI-LFD imaging can be accomplished with an endoscope, having a 305a multi-core fiber and/or a single-core fiber, or any multiple combination thereof, as light delivery mechanisms. , which is used with the volumetric imaging system (shown in Figure 1), according to any of the SVI techniques and the geometries and optical configurations described above. Typically, in an endoscopic imaging application, the various optical components and assemblies that are close to the sample are small in size, allowing for proximity delivery and collection of light to and from the sample.

Figure 3B shows an example of optical geometry for use with an endoscopic implementation of SVI-LFD imaging techniques. As illustrated in Figure 3B that an illumination light beam 306 is scanned by the scanning device 315 and optically coupled into different cores of a multi-core optical fiber, which is a part of an endoscope, using a 310 coupling lens. This beam of light is carried by the endoscope to a remote location that is otherwise inaccessible to some microscopy-based imaging techniques. Since the light beam is carried by multiple nuclei, the light beam exiting the endoscope and illuminating the sample scans or paints a selective 3D volume of the sample according to any of the SVI techniques described above. Some examples of scanning devices are galvanometers, resonant scanners, acousto-optic baffles, digital micromirror devices, which perform scanning in 1, 2, or 3 directions, or any combination of multiple directions.

Figure 3C shows an example of another optical geometry for use with an endoscopic implementation of SVI-LFD imaging techniques. In Figure 3C, a multicore fiber 305c is used for delivery of the excitation light from the endoscope 307 to the illuminated sample, and free space optics are employed for the light field detection aspects of the image. optical geometry. Light is illustrated as propagated through an illumination objective lens 320, and focused to a point and scanned in two dimensions, or to a line and scanned in one dimension on the proximal face of the fiber bundle. In some cases, the distal end, ie the end away from the sample, of the endoscope is attached to the microscope assembly of the volumetric imaging system (shown in Figure 1). In this case, both excitation and detection can be performed using a single objective lens, using an optical geometry that is similar to that depicted in Figure 2B, referring to the static optical volume approach for SVI. Figure 3C shows the use of two lenses, in the optical geometry configuration, the illumination objective lens 320 to receive the illumination light, and the detection objective lens 330 to receive the light field 335 for detection. In some implementations, the optical geometry shown in Figure 3C can be adjusted to arrange the two objective lenses 320, 330 at an angle 0 theta that is less than 90°. In another implementation, the optical geometry may be adjusted to include a miniature prism for rotating the illumination light 321 through an angle of 90° in order to arrange the illumination light path and the detection light path orthogonally between each other. yes, in order to achieve SVI techniques as described to optimize sample volume excitation.

Figure 3D shows an example of another optical geometry for use with an endoscopic implementation of SVI-LFD imaging techniques. In the example shown, a multicore fiber 305d is a delivery fiber for the endoscope 340, and is used to deliver excitation light 345 to a sample. Optical geometry is also shown using a 350 illumination objective lens and a 355 tube lens for creating an image, in accordance with the SVI-LFD imaging techniques disclosed herein. In some cases, the illumination objective lens is implemented as a miniature objective lens. The image is then transmitted to a distal face of endoscope 340 using another multicore fiber 305e. Similar to the optics of Figure 3C, the excitation light 345 is a beam that is focused to a point, and scanned in two dimensions, or to a line, and scanned in one dimension on the distal face of the fiber bundle. Figure 3D refers to the representation of two multi-core fibers 305d, 305e as being in the same endoscope 340. A detection objective lens 360 is illustrated which receives the light field that is propagated for detection as it is reflects, and is directed toward the other multicore fiber 305e of the endoscope 340 used for imaging. Alternatively, an implementation may include each of the multicore fibers 305d,305e being present in a different endoscope, and thus having two endoscopes separated in optical geometry. In this case, both the excitation and detection aspects of the SVI-FLD techniques can use a single objective lens, as discussed with reference to Figure 3C. Alternatively, an implementation can include multiple single-core fibers used for the excitation and sensing aspects of the SVI-LFD that are arranged in a single or multiple endoscopes. In another implementation, the optical geometry can be adjusted to include a miniature prism, similar to that discussed in reference to Figure 3C.

Figure 3E shows an example of another optical geometry for use with an endoscopic implementation of SVI-LFD imaging techniques. In this example, the endoscopy is performed using a single core fiber 365 to deliver excitation light from an endoscope 370, instead of a multicore fiber as shown in the optical geometries of Figure 3A and Figure 3A. 3D. Excitation light 375 is delivered as a beam, propagating through a lens 380, which is further scanned using a scanning device 385, which may be a Micro-Electro-Mechanical Systems (MEMS) mirror device. Figure 3E indicates that the scanning device 385 is configured to rotate in the x-y direction. Also, in some optical geometries, multiple scanning devices can be used. The light is then shown to be received by an illumination objective lens 390, and subsequently reflected in the direction for detection through the detection objective lens 395 and along the detection direction 396. In some implementations, two scanning devices may be used. A set of microlenses (not shown) may be located at the proximal end of the scope and is used to create the light field image, which is then transmitted to the distal face of the scope 370, for example, using another multiplex fiber. core. Thus, as an example, an optimal geometry for endoscope-based SVI may use a single core fiber, and a multicore fiber.

Figure 3F shows an example of an optical geometry in which fibers 400 and 401 are used to transmit illumination and detection light, respectively, and illumination and detection light are propagated through a common optical system 402, following the various implementations described above for the case where no fibers are used in the imaging apparatus. The optical fibers 401 and 402 can be multicore or single core, and together they form the endoscope 403 which allows SVI-LFD imaging of samples that would not otherwise be spatially accessible.

Figures 4A-4C show various examples of optical geometries for use with an SVI implementation using multi-view LFD imaging techniques. Each of the above approaches and implementations used to achieve the SVI-LFD imaging techniques disclosed herein have generally been described as imaging the sample using one direction for detection, thereby capturing the LFD image data from a single point of view. However, multi-view LFD imaging involves the use of various optical geometries that allow a sensing light field to be captured from multiple directions and/or views, effectively resulting in imaging of the sample volume from more than one direction. As an example, an optical geometry capable of supporting multi-view LFD imaging may include the use of multiple pairs of substantially orthogonal objective lenses for illumination and detection to image the 3D sample volume, in a manner somewhat similar to that of the optical geometry shown in Figure 2A (using a single lens for illumination and detection). SVI is achieved in each of these multiple-view imaging cases. Consequently, multi-view LFD imaging techniques can offer several advantages associated with imaging a volume of interest in 3D using multiple and varied perspectives. For example, computational reconstruction of LFD images that are generated from more than one direction according to multiple view techniques, can later be combined to obtain a final image with higher resolution and/or better spatial coverage.

In some implementations imaging with more than one view of the sample may be accomplished by sequentially rotating the sample through the multiple views; or/and using more than one pair of illumination-detection optical systems, oriented to look at the sample through different views, and the different views are photographed simultaneously or sequentially, without the need to physically rotate the sample. the sets The resulting image data from the multiple views could then be combined and reconstructed to obtain a final image with higher resolution and/or better spatial coverage.

Figure 4A shows an optical geometry for use with an SVI implementation using multi-view LFD imaging techniques. As illustrated in Figure 4A that multi-view optics involves the use of a single primary objective lens 405, and more than one set 410a, 410b of detection microlenses. By positioning the array pair 410a, 410b of detection microlenses, each with its optical axis making a finite angle with the optical axis of the primary microscope objective 405, the field of light 415 can be captured from multiple views, thus obtaining Images of the sample volume from multiple directions. Furthermore, the illumination light 420 propagates along more than one illumination axis. Illumination light 420 is then reflected using a mirror 425, to be directed towards the sample, thus creating more than one imaging volume 430a, 430b associated with a different view. Accordingly, the light field 415 captured from the volumes 430a, 430b illuminated by each microlens array 410a, 410b is further captured using cameras in order to generate LFD images corresponding to the respective view. In some embodiments, more than two sets of microlenses may be used in the optical geometry, such that it covers the entire angular range of the light field space. The multi-view light field imaging described herein, with multi-detection microlens arrays and cameras, achieves a detection rate equivalent to light field capture of the sample with a larger numerical aperture and a larger camera sensor area (than can be achieved with a single set of microlenses and a single camera).

In some cases, the number of sensing optics (corresponding to the number of image views) implemented using the optical configuration of Figure 4A, is a tunable characteristic that can be adjusted to achieve a desirable result. Increasing the number of sensing elements used in the optical arrangement can be a tradeoff. For example, more sets of microlenses used can capture more angular views, but can potentially decrease the numerical aperture (and resolution) of each set, while the final reconstructed resolution can depend on both the number of views and the intrinsic resolution. achieved in each view. In some scenarios, determining the number of microlens arrays used, for example, may depend on imaging variables such as the desired resolution and the field of view of the given 3D sample. Light field detection with a substantially large number of microlens arrays oriented at finite angles to the main optical axis allows the capture of high numerical apertures through the primary microscope objective (i.e., more angular information), but without the penalty of the reduction of the axial coverage (z axis).

The optical geometry depicted in Figure 4A used for multi-view LFD imaging can also be tuned to provide SVI techniques. For example, illumination light 420 is illustrated as being delivered parallel to the principal optical axis of microscope primary objective 405, adjusted to cover, in the xy plane, the desired xy cross section of the volume of interest. Furthermore, the illumination light 420 is directed along the central optical axis of the microscope objective, and the detection path is along an oblique angle direction of the microscope objective 405. The oblique angled nature of the detection path allows multiple detection paths to be performed simultaneously, or sequentially, through the same microscope objective 420, without the need to physically rotate the sample. Also, since the detection volumes are angled to the main optical axis, SVI is achieved. In some cases, the optics can be properly adjusted by allowing the detection volumes (detection) to be orthogonal to the main optical axis (illumination). In some implementations, the 3D sample volume is selected for SVI techniques using linear optical excitation. In some implementations, SVI techniques are achieved using multi-photon nonlinear optical excitation. Figure 4A depicts two oblique angle detection views, but more views can be performed to cover more of the angular range of light field which is coming from the sample. For example, on the image side of microscope objective 405, beam splitters of various types (eg, dichroic or partially reflective) can be used to separate illumination and detection paths in different directions. Multiple detection views are recorded in various ways, for example, with multiple arrays 410a, 410b of microlenses and camera optics oriented at the appropriate angles, as depicted in Figure 4A. In some cases, multiple slant-angle illumination direction paths may be used to provide the SVI for the multiple slant-angle detection paths.

Another example of an optical geometry for use with an SVI implementation using multi-view LFD imaging techniques is shown in Figure 4B, where region overlap occurs between multiple image views. The shaded region 435 represents an overlap between the detected volumes with corresponding image views, ie, volume 440a from a first image view and volume 440b from a second image view. Consequently, there is an imaging benefit of multi-view LFD, as computational reconstruction of the two rendered views (which have a shared imaging volume that produces more image data associated with that particular volume) can provide a final 3D image of volume 435 superimposed with higher resolution.

Also, the lighting shown in Figure 4B includes multiple side lighting 445a, 445b located on either side (i.e., to the left, right) of, and angling to the axis of, a central lighting 445c. . Furthermore, the side illuminations 445a, 445b are illuminations at an oblique angle with respect to the optical axis of the microscope objective. The central illumination 445c is an illumination that is directed along the central axis of the microscope objective. The optical geometry of Figure 4B is configured such that a right side illumination 445b enables SVI for detection of the view associated with volume 440b, while a left side illumination 445a enables SVI for volume 440a of the other view. Thus, LFD image acquisition may sequentially involve imaging one view associated with right side illumination 445b and then imaging another view associated with left side illumination 445a. Consequently, each view of the multi-view LFD images performed can have an optimal high-contrast SVI. In some implementations, multiple view imaging techniques can decrease the imaging rate associated with the procedure by a rate related to the number of views. For example, the example shown in Figure 4B using two image views can decrease the imaging rate by a factor of two. This can be beneficial in situations where high-speed imaging is not as desirable as high-resolution imaging.

Figure 4C shows an example of another optical geometry for multiple view LFD imaging techniques, in which the optical paths associated with each of the multiple imaging views are separated by optics. In Figure 2C, an optical element, shown as a reflective (mirrored) prism 450, is included in the optical geometry for manipulating light in a manner that separates the optical paths for different image views. Reflective prism 450 is illustrated as being positioned on the image side of microscope primary objective lens 455 . Illumination light 460a, 460b is emitted toward reflective prism 450. In this case, the illumination is directed to two opposite sides of the reflective prism 450, representative of a corresponding imaging viewpoint based on each illumination side. Illumination light 460a (left side) and 460b (right side) is then reflected, or otherwise redirected, by the surface of prism 450 (at a right angle), on both sides, having two separate illumination paths. Each distinct illumination path spreads the illumination light 460a, 460b through the primary objective lens 455 to create multiple illuminated volumes of the sample, corresponding to a respective image view 465a, 465b.

Additionally, the light fields 462a, 462b are shown to have separate optical paths, due to the reflective prism 450. Therefore, the optical geometry allows detecting, using microlens arrays, for example, the light field 462a of one image view and the light field 462b of another image view at separate locations. In some cases, this optical arrangement provides more space, for example, to implement a camera detecting the light field captured by each array of microlenses. In some implementations, the optics used to arrange the separate optical paths is another optical element with reflective properties, such as a dichroic mirror that transmits the illumination light and reflects the detected light. In this case, the illumination light can be delivered along the optical axis of the primary microscope objective 455 . Although Figure 4C shows an example of an optical geometry that uses two reflective surfaces to redirect the different image views, more than two surfaces (more than two illumination and detection paths) may be used in some implementations in order to cover everything. the angular interval of return light field in the microscope objective lens 455.

Other types of lighting mechanisms may also be used in each of the various approaches and implementations described herein above, to adequately achieve the SVI aspects of the embodiments. In various implementations, volume-selective illumination is achieved by manipulating a coherent nature of the illumination light, which is characteristic of some light emission technologies, such as holography, interferometry, and the like. Consequently, coherent light sources can be used for illumination of a sample in any of the disclosed SVI-LFD imaging embodiments, to achieve specific spatial regions of constructive and destructive light interference, thus providing selective illumination in the volume of interest desired. In some implementations, coherent light techniques can generate image contrast using linear optical excitation, or multi-photon nonlinear optical excitation.

Embodiments of subject matter and functional operations described herein may be implemented in digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed herein and their structural equivalents, or in combinations of one or more of these. Embodiments of the subject matter described herein may be implemented using one or more computer program instruction modules encoded on a computer readable medium for execution by, or to control the operation of, a data processing apparatus. data. The computer-readable medium can be a manufactured product, such as a hard drive in a computer system or an optical disc sold through retail channels, or an embedded system. The middle computer-readable may be purchased separately and subsequently encoded with one or more computer program instruction modules, such as by delivering one or more computer program instruction modules over a wired or wireless network. The computer-readable medium can be a machine-readable storage device, a machine-readable storage substrate, a memory device, or a combination of one or more of these.

The term "data processing apparatus" encompasses all apparatus, devices, and machines for data processing, including by way of example a programmable processor, a computer, or multiple processors or computers. The apparatus may include, in addition to hardware, code that creates an execution environment for the computer program in question, for example, code that constitutes the firmware of the processor, a protocol stack, a database management system, an operating system, a runtime environment, or a combination of one or more of these. In addition, the apparatus may employ various infrastructures of different computing models, such as web services, distributed computing, and network computing infrastructures.

A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and can be deployed in any form. , including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program does not necessarily correspond to a file on a file system. A program can be stored in a portion of a file that contains other programs or data (for example, one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in multiple coordinated files ( for example, files that store one or more modules, subprograms, or pieces of code). A computer program can be deployed to run on one computer or on multiple computers located at one site or distributed over multiple sites and interconnected by a communication network.

The procedures and logic flows described herein may be performed by one or more programmable processors executing one or more computer programs to perform functions by operating on input data and generating output. The logic procedures and flows may also be realized by, and the apparatus may also be implemented as, special purpose logic circuits, for example, an FPGA (Field Programmable Gate Assembly) or an ASIC (Application Specific Integrated Circuit).

Processors suitable for executing a computer program include, by way of example, general purpose and special purpose microprocessors, and any one or more processors of any type of digital computer. In general, a processor receives instructions and data from read-only memory, random-access memory, or both. The essential elements of a computer are a processor to carry out instructions and one or more memory devices to store instructions and data. In general, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, for example, magnetic, magneto-optical, or optical disks. However, it is not necessary for a computer to have such devices. In addition, a computer may be embedded in another device, such as a mobile phone, personal digital assistant (PDA), mobile audio or video player, game console, Global Positioning System (GPS) receiver, etc. or a portable storage device (for example, a Universal Serial Bus (USB) flash drive), to name just a few. Devices suitable for storing computer program instructions and data include all forms of non-volatile memory, memory media, and memory devices, including, by way of example, semiconductor memory devices, for example, EPROM (Programmable Read Only Memory). and Erasable), EEPROM (Electrically Erasable Programmable Read-Only Memory), and flash memory devices; magnetic disks, for example, internal hard drives or removable disks; magneto-optical disks; and CD-ROM and DVD-ROM discs. The processor and memory may be supplemented by, or incorporated into, special purpose logic circuits.

To provide interaction with a user, embodiments of the subject matter described herein may be implemented in a computer having a display device, for example, LCD (liquid crystal display), OLED (light emitting diode organic) or other monitor, for displaying information to the user, and a keyboard and pointing device, eg, a mouse or trackball, by which the user can provide input to the computer. Other types of devices may also be used to provide interaction with a user; For example, the feedback provided to the user can be any form of sensory feedback, eg, visual, auditory, or tactile feedback; and user input can be received in any form, including acoustic, verbal, or tactile input.

The computer system may include clients and servers. A client and a server are generally remote from each other and typically interact through a communication network. The client and server relationship arises by virtue of that the computer programs run on the respective computers and have a client-server relationship with each other. Embodiments of the subject matter described herein may be implemented in a computer system that includes a back-end component, for example, such as a data server, or that includes a middleware component, for example, a application server, or including a frontend component, for example, a client computer having a graphical user interface or a web browser through which a user can interact with an implementation of the subject matter described herein description, or any combination of one or more such back-end, middleware, or front-end components. System components may be interconnected by any form or means of digital data communication, eg, a communication network. Examples of communication networks include a local area network ("LAN") and a wide area network ("WAN"), an internetwork (for example, the Internet), and peer-to-peer networks (for example, ad hoc networks between peers).

Although this specification contains many implementation details, these should not be construed as limiting the scope of the invention or what can be claimed, but rather as descriptions of specific features of particular embodiments of the invention. Certain features that are described herein in the context of separate embodiments may also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment may also be implemented in multiple embodiments separately or in any suitable sub-combination. Furthermore, while features may be described above as acting in particular combinations and even initially claimed as such, one or more features of a claimed combination may in some cases be eliminated from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

Similarly, although operations are shown in the drawings in a particular order, this is not to be construed as requiring such operations to be performed in the particular order shown or in sequential order, or that all operations illustrated be performed. to achieve the desired results. Under certain circumstances, multitasking and parallel processing can be advantageous. In addition, the separation of various system components in the embodiments described above is not to be construed as requiring such separation in all embodiments, and it is to be understood that the program components and systems described can generally be integrated together into a single product of software or packaged into multiple software products.

Therefore, particular embodiments of the invention have been described. Other embodiments are within the scope of the following claims. Furthermore, the actions mentioned in the claims can be performed in a different order and still achieve the desired results.

Claims (11)

A volumetric imaging apparatus (105) comprising: a light source (106) configured to emit illumination light propagating through an illumination light path to illuminate a three-dimensional (3D) sample (126); and an optical system (110) arranged with respect to the light source (106) to receive a light field, which comes from the illuminated 3D sample (126), wherein the light field propagates through a detection light path; wherein the light source (106), the optical system (110), or both, are configurable to perform selective volume illumination, which selects a volume for 3D confined illumination of the 3D specimen (126). based on the 3D sample (126) to be illuminated and a light field detection (LFD) method to be applied, wherein the 3D confined illumination is a selected volume of the sample (126 ) in 3D that will be particularly excited by an illumination light for imaging wherein the apparatus (105) optionally comprises an endoscope (340), and wherein the detection light path is not collinear to the illumination light path, and the 3D confined illumination volume of the 3D specimen is selected by adjusting the 3D optical geometry of the illumination light relative to the optical system, and in which a) or b), in which a) the optical system (110) comprises a first optical element arranged to receive illumination light propagating through the illumination light path to illuminate the sample in 3D (126), and a second optical element arranged to receive the light field propagating through the illumination light path. detection light path which comes from the illuminated 3D sample (126), wherein the first optical element and the second optical element are arranged to adjust the 3D optical geometry of the illumination light path at an angle 0 with the detection light path that is greater than zero to select the volume of the 3D confined illumination of the sample (126) in 3D, or b) wherein the optical system comprises a common optical element arranged to receive illumination light propagating through the illumination light path to illuminate the sample in 3D, receiving the light field propagating through the path of detection light which comes from the illuminated 3D sample, and adjusting the 3D optical geometry of the light source to select the volume of the 3D confined illumination of the 3D sample, and either i) or ii) , in which i) wherein the volume of the 3D confined illumination of the 3D sample (126) is selected using a focused beamform of the illumination light, and a 3D optical geometry of the light source adjusted to illuminating multiple subregions of the volume of the 3D confined illumination of the sample (126) in 3D in one scan through the entirety of the selected 3D confined illumination of the sample (126) in 3D during one exposure time, or ii) wherein the illumination light can be used to change the photochemical and physical properties of one or more contrast agents in the sample (126) in 3D, offering enhanced contrast in the 3D confined illumination volume of the sample. displays (126) in 3D, and an additional illumination light source (106) and associated optical system (110) for providing light field images of the enhanced contrast agents. The apparatus (105) of claim 1, wherein the volume of 3D confined illumination of the 3D sample is selected using linear optical excitation. The apparatus (105) of claim 1, wherein the volume of 3D confined illumination of the 3D sample (126) is selected using multi-photon nonlinear optical excitation and fitting a 3D optical geometry of the light source (106) in relation to the optical system (110). The apparatus (105) of claim 1, wherein the volume of the 3D confined illumination of the sample (126) is selected using direct beamforming of the illumination to produce a spatially extended volume of light. . The apparatus (105) of claim 1, comprising switchable optical components and optical geometries, the apparatus being a plug-in module configured to fit an existing microscope, or being an integrated module of a microscope designed from scratch said microscope having multiple imaging capabilities, wherein the multiple imaging capabilities comprise performing high resolution 3D optical sectioning with submicron to 2 micron resolution. The apparatus (105) of claim 1, option b), wherein the illumination light propagates along an oblique angle path through the common optical element, and the detection light path is collinear. with the optical axis of the common optical element, making an angle theta that is greater than 0 with the illumination light path. 7. A volumetric imaging system comprising: the apparatus (105) of any one of claims 1 and 3, an LFD detection mechanism arranged with respect to the apparatus (105) to receive the light field, which comes from the illuminated 3D sample (126), and to capture the LFD images projected by the light field; a computer communicatively coupled with the LFD detection mechanism and configured to generate volumetric imaging data of the sample (126) in 3D by performing a computational reconstruction of the LFD images. The apparatus (105) of claim 1, wherein the volume of 3D confined illumination is selected by illuminating the 3D sample (126) in a patterned or structured manner that results in a detected light field that covers a desired 3D space of the sample (126) in 3D, the usable patterns or structures to allow a computationally optimized reconstruction of the final image data from the detected light fields. The apparatus (105) of claim 1, wherein the optical system (110) comprises a plurality of optical elements arranged to adjust 3D optical geometry to create a plurality of sensing light paths, each at a respective angle to the illumination light path, and usable to (i) select the 3D confined illumination volume of the sample 126 in 3D, (ii) receive the light field propagating through the detection light path from multiple views associated with the respective angles, and (iii) computational reconstruction of the LFD images from the multiple views. The apparatus (105) of claim 1, wherein the volume of 3D confined illumination of the 3D sample (126) is selected by adjusting the optical system (110) using (i) a single core fiber to emitting the illumination light that propagates through the illumination light path to illuminate the sample (126) in 3D, or (ii) a multi-core fiber to emit the illumination light that propagates through the light path to illuminate the sample (126) in 3D, or (iii) a combination of one or more single-core fibers and multi-core fibers to emit the illumination light that propagates through the illumination light path to illuminate the sample (126) in 3D. The apparatus (105) of claim 1, wherein the volume of 3D confined illumination of the 3D sample (126) is selected by adjusting the optical system (110) using (i) a single core fiber to receive the light field which comes from the illuminated 3D display (126), or (ii) a multicore fiber to receive the light field which comes from the illuminated 3D display (126), or (iii) a combination of one or more of the single-core fibers and the multi-core fibers to receive the light field which comes from the illuminated 3D display (126).